Use of isolated domains of type IV collagen to modify cell and tissue interaction

ABSTRACT

The instant invention provides methods and kits for inhibiting angiogenesis, tumor growth and metastasis, and endothelial cell interactions with the extracellular matrix, involving contacting the tumor or animal tissue with at least one isolated type IV collagen NCl α chain monomer. In a specific embodiment of the invention, the isolated domain of type IV collagen comprises the NCl (α1), (α2), (α3), or (α6) chain monomer, or protein constructs having substantially the same structure as the NCl (α1), (α2), (α3), or (α6) chain monomer.

CROSS REFERENCE

[0001] The present application is a continuation in part of U.S. PatentApplications S No. 60/106,170 filed Oct. 29, 1998; 60/079,783 filed Mar.27, 1998; and Ser. No. 09/183,548 filed Oct. 30, 1998, which is acontinuation of U.S. application Ser. No.08/800,965 filed Feb. 18, 1997,now U.S. Pat. No. 5,856,184, which is a continuation of U.S. applicationSer. No. 08/497,206 filed Jun. 30, 1995 now U.S. Pat. No. 5,691,182, allof which are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] This invention relates to methods and kits for inhibitingangiogenesis, tumor growth and metastasis, and endothelial cellinteractions with the extracellular matrix.

BACKGROUND OF THE INVENTION

[0003] Angiogenesis, the process of formation of new blood vessels,plays an important role in physiological processes such as embryonic andpostnatal development as well as in wound repair. Formation of bloodvessels can also be induced by pathological processes involvinginflammation (e.g., diabetic retinopathy and arthritis) or neoplasia(e.g., cancer) (Folkman, 1985, Perspect, Biol. Med., 29, 10).Neovascularization is regulated by angiogenic growth factors secreted bytumor or normal cells as well as the composition of the extracellularmatrix and by the activity of endothelial enzymes (Nicosia andOttinetti, 1990, Lab. Invest., 63, 115).

[0004] During the initial stages of angiogenesis, endothelial cellsprouts appear through gaps in the basement membrane of pre-existingblood vessels (Nicosia and Ottinetti, 1990, supra; Schoefl, 1963,Virehous Arch, Pathol. Anat. 337, 97-141; Ausprunk and Folkman, 1977,Microvasc. Res. 14, 53-65; Paku and Paweletz, 1991, Lab. Invest. 63,334-346). As new vessels form, their basement membrane undergoes complexstructural and compositional changes that are believed to affect theangiogenic response (Nicosia, et. al., 1994, Exp. Biology, 164,197-206). Early planar culture models have shown that basement membranemolecules modulate the attachment, migration and proliferation andorganizational behavior of endothelial cells (Nicosia, et. al., 1994,supra). More recent studies with three-dimensional aortic culture modelsthat more closely simulate angiogenic conditions during wound healing invivo suggest that basement membrane is a dynamic regulator ofangiogenesis whose function varies according to its molecular components(Nicosia, 1994, supra).

[0005] A common feature of all solid tumor growth is the requirement fora blood supply. Therefore, numerous laboratories have focused ondeveloping anti-angiogenic compounds based on growth factors and theirreceptors. While this approach has led to some success, the number ofgrowth factors known to play a role an angiogenesis is large. Therefore,the possibility exists that growth factor antagonists may have onlylimited use in treating cancer since tumors and associated inflammatorycells likely produce a wide variety of factors that can induceangiogenesis.

[0006] In this regard, a strategy that targets a common feature ofangiogenesis, such as endothelial cell adhesion to the extracellularmatrix (ECM), might be expected to have a profound physiological impacton tumor growth in humans. This notion is supported by the fact thatantagonists of specific ECM cell adhesion receptors such as αvβ3 andαvβ5 integrins can block angiogenesis. Furthermore, the αvβB3 integrinis expressed most prominently on cytokine-activated endothelial andsmooth muscle cells and has been shown to be required for angiogenesis.(Varner et al., Cell Adhesion and Communication 3:367-374 (1995); Brookset al., Science 264:569-571 (1994)). Based on these findings, apotentially powerful new approach to anti-angiogenic therapy might be tospecifically target critical regulatory domains within distinct ECMcomponents.

[0007] The basement membrane (basal lamina) is a sheet-likeextracellular matrix (ECM), which is a basic component of all tissues.The basal lamina provides for the compartmentalization of tissues, andacts as a filter for substances traveling between tissue compartments.Typically the basal lamina is found closely associated with anepithelium or endothelium in all tissues of an animal including bloodvessels and capillaries. The basal lamina components are secreted bycells and then self assemble to form an intricate extra-cellularnetwork. The formation of biologically active basal lamina is importantto the development and differentiation of the associated cells.

[0008] Type IV collagen has been shown to be a major structuralcomponent of basement membranes. The protomeric form of type IV collagenis formed as a heterotrimer made up from a number of different subunitchains called α1(IV) through α6(IV). Up to now, six genetically distinctα-chains belonging to two classes with extensive homology have beenidentified, and their relative abundance has been demonstrated to betissue specific. The type IV collagen heterotrimer is characterized bythree distinct structural domains: the non-collagenous (NCl) domain atthe carboxyl terminus; the triple helical collagenous domain in themiddle region; and the 7S collagenous domain at the amino terminus.(Martin, et. al., 1988, Adv. Protein Chem. 39:1-50; Gunwar, et. al.1991, J. Biol. Chem. 266:14088-14094).

[0009] The capability of expression of recombinant α(IV) NCl domainsprovides the opportunity to study the effect of specific domains on manybiological processes, such as angiogenesis, tumor metastasis, cellbinding to basement membranes, and assembly of Type IV collagenmolecules.

SUMMARY OF THE INVENTION

[0010] The instant invention provides methods and kits for inhibitingangiogenesis, tumor growth and metastasis, and endothelial cellinteraction with the extracellular matrix, each method comprisingcontacting the tumor or animal tissue with an one or more isolated typeIV collagen NCl α chain monomer selected from the group consisting ofα1, α2, α3, and α6 NCl chain monomers.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 illustrates the effects of NCl (Hexamer) and 7S domains ofType IV collagen at a 50 μg/ml concentration on angiogenesis from mousethoracic aorta organ cultures.

[0012]FIG. 2 illustrates the effects of 7S domain of Type IV collagen onangiogenesis from mouse thoracic aorta organ cultures. The domainconcentrations employed in this experiment were 0 μg/ml (control); 0.5μg/ml; 5 μg/ml and 50 μg/ml.

[0013]FIG. 3 illustrates the effects of NCl (Hexamer) domain of Type IVcollagen on angiogenesis from mouse thoracic aorta organ cultures. Thedomain concentrations employed in this experiment were 0 μg/ml(control); 5 μg/ml and 5 μg/ml and 50 μg/ml.

[0014]FIG. 4 are photographs of mouse thoracic aorta segments embeddedin Matrigel (EHS basement membrane matrix, Collaborative BiomedicalProducts, Bedford, Mass.) at 5 days of culture. Control specimen (0μg/ml of NCl (Hexamer) and 7S domains) exhibited growth of microvesselsfrom the cultured tissue into the matrix (FIG. 4A). In contrast,angiogenesis was inhibited in specimens cultured with 50 μg/ml of 7Sdomain (FIG. 4B) and NCl (Hexamer) domain (FIG. 4C).

[0015]FIG. 5 is a graphical representation of data demonstrating the invivo effect of IV injection of recombinant (α1) type IV collagen monomeron angiogenesis using fibrin implants in rats.

[0016]FIG. 6 is a graphical representation of data demonstrating thatthe recombinant (α1) and (α2) NCl monomers inhibit the bFGF-inducedincrease in angiogenic index in vivo.

[0017]FIG. 7 is a graphical representation of demonstrating the doseresponse effect of recombinant (α2) NCl monomer on the bFGF-inducedincrease in total blood vessel branch points in vivo.

[0018]FIG. 8 is a graphical representation of data demonstrating thedose response effect of recombinant (α2) NCl monomer on the bFGF-inducedincrease in angiogenic index in vivo.

[0019]FIG. 9 is a graphical representation of data demonstrating thedose response effect of recombinant (α2) NCl monomer on the bFGF-inducedincrease in angiogenic index in vivo.

[0020]FIG. 10 is a graphical representation of data demonstrating theeffect of recombinant (α1) and (α2) NCl monomers on mean CS-1 melanomatumor weight in vivo.

[0021]FIG. 11 is a graphical representation of data demonstrating thedose response effect of recombinant (α2) NCl monomer on mean CS-1melanoma tumor weight in vivo.

[0022]FIG. 12 is a graphical representation of data demonstrating theeffect of recombinant (α1), (α2), and (α4) NCl monomers on mean HT1080tumor weight in vivo.

[0023]FIG. 13 is a graphical representation of data demonstrating theeffect of recombinant (α1), (α2), (α3) and (α5) NCl monomers on meanHEP-3 tumor weight in vivo.

[0024]FIG. 14 is a graphical representation of data demonstrating humanendothelial cell adhesion to immobilized NCl α monomers.

[0025]FIG. 15 is a graphical representation of data demonstrating theeffect of soluble α1 and α2 NCl monomers on human endothelial celladhesion to pepsinized collagen type IV.

[0026]FIG. 16 is a graphical representation of data demonstrating theeffect of isolated recombinant NCl monomers on human endothelial cellmigration in vitro.

[0027] FIGS. 17A-F provides the sequences of each type IV collagen achain monomer.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Within this application, unless otherwise stated, the techniquesutilized may be found in any of several well-known references such as:Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, ColdSpring Harbor Laboratory Press), Gene Expression Technology (Methods inEnzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, SanDiego, Calif.), “Guide to Protein Purification” in Methods in Enzymology(M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: AGuide to Methods and Applications (Innis, et al. 1990. Academic Press,San Diego, Calif.), Culture of Animal Cells: A Manual of BasicTechnique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.),and Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J.Murray, The Humana Press Inc., Clifton, N.J.).

[0029] As used herein, the term Type IV collagen domain encompasses thegroup of molecules including the non-collagenous NCl domain (Hexamer)and 7S collagenous domains, as well as NCl α chain monomers.

[0030] The invention comprises methods for using Type IV collagen NClα-monomers (ie: α1, α2, α3, and α6), which are defined to include suchmonomers isolated from any multicellular organism or produced viarecombinant protein expression from a gene encoding such a monomer fromany multicellular organism, and also to encompass various modifications,additions, and/or deletions to such monomers.

[0031] In one aspect, the present invention provides methods and kitsfor inhibiting angiogenesis in an animal tissue comprising contactingthe tumor or animal tissue with an amount effective to inhibitangiogenesis of a polypeptide composition comprising one or moreisolated type IV collagen NCl α chain monomer selected from the groupconsisting of α1, α2, α3, and α6 NCl chain monomers.

[0032] In another aspect, the present invention provides methods andkits for inhibiting tumor growth in tissue comprising contacting thetumor or tissue with an amount effective to inhibit tumor growth of apolypeptide composition comprising one or more isolated type IV collagenNCl α chain monomer selected from the group consisting of α1, α2, α3,and α6 NCl chain monomers.

[0033] In another aspect, the present invention provides methods andkits for inhibiting tumor metastasis in tissue comprising contacting thetumor or tissue with an amount effective to inhibit metastasis of apolypeptide composition comprising one or more isolated type IV collagenNCl α chain monomer selected from the group consisting of α1, α2, α3,and α6 NCl chain monomers.

[0034] In a further aspect, the present invention provides methods andkits for inhibiting endothelial cell interactions with the extracellularmatrix in tissue comprising contacting the tumor or tissue with anamount effective to inhibit endothelial cell interactions with theextracellular matrix of a polypeptide composition comprising one or moreisolated type IV collagen NCl α chain monomer selected from the groupconsisting of α1, α2, α3, and α6 NCl chain monomers.

[0035] The NC1-encoding domain of each of the six a chain cDNAs has beencloned into a vector for recombinant protein expression as previouslydescribed (Sado et al., Kidney Intl. 53:664-671 (1998), incorporated byreference herein in its entirety). The vectors are used to stablytransfect human kidney 293 cells, which produce the recombinant protein.The DNA and deduced amino acid sequences of the recombinant type IVcollagen alpha chain monomers produced as described are shown in FIGS.17A-F. The first 17 amino acids corresponds to a BM40 signal sequence(which is cleaved from the mature protein), to facilitate proteinsecretion. All the secreted proteins (ie: mature proteins) start withthe sequence APLA followed by the affinity tag, DYKDDDDK at the aminoterminus. This tag facilitates purification and identification of thematerial, and does not interfere with biological activity of therecombinant NCl α chain monomer.

[0036] The type IV collagen NCl α chain monomers can be produced by anymethod known in the art, including using recombinant DNA technology orbiochemical peptide synthesis technology, or by isolating the NCldomains from animal sources, such as from basement membrane sources suchas bovine lens capsule and bovine kidney glomeruli. (Peczon et al., Exp.Eye Res. 30:155-165 (1980); Langeveld et al., J. Biol. Chem.263:10481-10488 (1988); Gunwar et al., J. Biol. Chem. 266:14088-14094(1991))

[0037] In practicing the invention, the amount or dosage range of typeIV collagen NCl α chain monomers employed is one that effectivelyinhibits angiogenesis, tumor growth, tumor metastasis, and/orendothelial cell-extracellular matrix interactions. An inhibiting amountof NCl α chain monomers that can be employed ranges generally betweenabout 0.01 μg/kg body weight and about 10 mg/kg body weight, preferablyranging between about 0.05 μg/kg and about 5 mg/kg body weight.

[0038] The NCl α chain monomers may be administered by any suitableroute, including orally, parentally, by inhalation spray, rectally, ortopically in dosage unit formulations containing conventionalpharmaceutically acceptable carriers, adjuvants, and vehicles. The termparenteral as used herein includes, subcutaneous, intravenous,intraarterial, intramuscular, intrasternal, intratendinous, intraspinal,intracranial, intrathoracic, infusion techniques or intraperitoneally.In preferred embodiments, the NCl α chain monomers are administeredintravenously or subcutaneously.

[0039] The NCl α chain monomers may be made up in a solid form(including granules, powders or suppositories) or in a liquid form(e.g., solutions, suspensions, or emulsions). The NCl α chain monomersof the invention may be applied in a variety of solutions. Suitablesolutions for use in accordance with the invention are sterile, dissolvesufficient amounts of the NCl α a chain monomers, and are not harmfulfor the proposed application.

[0040] The NCl α chain monomers may be subjected to conventionalpharmaceutical operations such as sterilization and/or may containconventional adjuvants, such as preservatives, stabilizers, wettingagents, emulsifiers, buffers etc.

[0041] For administration, the NCl α chain monomers are ordinarilycombined with one or more adjuvants appropriate for the indicated routeof administration. The compounds may be admixed with lactose, sucrose,starch powder, cellulose esters of alkanoic acids, stearic acid, talc,magnesium stearate, magnesium oxide, sodium and calcium salts ofphosphoric and sulphuric acids, acacia, gelatin, sodium alginate,polyvinylpyrrolidine, and/or polyvinyl alcohol, and tableted orencapsulated for conventional administration. Alternatively, thecompounds of this invention may be dissolved in saline, water,polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidalsolutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil,tragacanth gum, and/or various buffers. Other adjuvants and modes ofadministration are well known in the pharmaceutical art. The carrier ordiluent may include time delay material, such as glyceryl monostearateor glyceryl distearate alone or with a wax, or other materials wellknown in the art.

[0042] The present invention may be better understood with reference tothe accompanying examples that are intended for purposes of illustrationonly and should not be construed to limit the scope of the invention, asdefined by the claims appended hereto.

EXAMPLE 1 In Vitro Effect on Angiogenesis

[0043] With modifications, the procedures of Nicosia and Ottinetti(1990), supra, and Nicosia, et. al. (1994), supra, were utilized forexperiments designed to test the effect of Type IV collagen onangiogenesis under in vitro conditions. The model has been used to studythe effects of growth factors and extracellular matrix molecules on theangiogenic response and employs aortic rings cultures inthree-dimensional collagen gels under serum-free conditions. Theseexperiments are outlined below.

[0044] A. Methods

[0045] Experiments were performed with 1-3 month old Swiss Webster malemice. Following anesthesia, the thoracic aorta was excised under asepticconditions and transferred to sterile MCDB 131 sterile growth medium(Clonetics, San Diego, Calif.) containing antibiotics. Fat was dissectedaway from the aorta and approximately six to eight 1 mm thoracicsegments were obtained from each specimen. Segments were transferred to48 well tissue culture plates. The wells of these plates were layeredwith 100 microliters of Matrigel (EHS basement membrane, CollaborativeBiomedical Products, Bedford, Mass.) prior to transfer of the aorticsegments. The Matrigel was diluted 1:1 with MCDB 131 growth medium priorto use. The segments were centered in the wells and an additional 100microliters of Matrigel was then placed over the specimens. The aorticsegments were therefore embedded in the basement membrane matrix. Eachwell then received 300 microliters of MCDB 131 growth medium. The plateswere placed in an incubator maintained at 37° C. with 5% CO₂. Specimenswere observed daily over a 7 day period. Newly growing microvessels werecounted using an inverted phase microscope at various times during theculture period, but data is expressed at 3 and 5 days of culture. Totest for the effect of Type IV collagen on angiogenesis, domains atknown concentrations were mixed with the Matrigel and with the MCDB 131growth medium. Fresh MCDB 131 growth medium (plus and minus collagendomains) was changed every 3 days.

[0046] B. Results

[0047] After establishing the time course of angiogenesis under controlconditions (Matrigel plus MCDB 131 growth medium), experiments wereperformed using various concentrations of Type IV collagen (isolatedfrom bovine lens) NCl (hexamer) and 7S domains. Data represents theanalysis of at least 3 specimens per experimental condition. In thefirst experiment (FIG. 1), analysis indicated that at a concentration of50 μg/ml, NCl domain and 7S domain significantly inhibited angiogenesisas monitored at 3 and 5 days of culture. In the second experiment,various concentrations of these domains were analyzed. As indicated inFIG. 3, 7S domain at 50 μg/ml again significantly inhibited angiogenesisat 3 and 5 days. Inhibition was reduced at 5 and 0.5 μg/mlconcentrations. As indicated in FIG. 2, NCl domain was less effective inblocking angiogenesis as compared to that observed in the firstexperiment (FIG. 1), although it was still effective. In addition, ascompared to the 7S domain, there was less of a correlation betweenconcentration and inhibitory action.

[0048] FIGS. 4A-C are photographs of mouse thoracic aorta segmentsembedded in Matrigel (EHS basement membrane matrix, CollaborativeBiomedical Products, Bedford, Mass.) at 5 days of culture in thepresence or absence of 50 μg/ml of Type IV collagen domains. The controlspecimen (no domains) exhibited growth of microvessels from the culturedtissue into the matrix (FIG. 4A). In contrast, angiogenesis inhibitionwas observed in tissues cultured in the presence of 50 μg/ml of 7S (FIG.4B) and NCl (Hexamer) domain (FIG. 4C).

EXAMPLE 2 Subcutaneous Fibrin Implant Angiogenesis

[0049] Recombinant human type IV collagen NCl (α3) monomer (Sado et al.,Kidney International 53:664-671 (1998)) was injected intravenously inFisher 344 rats containing fibrin implants surgically placedsubcutaneously, a modified version of the method described by Dvorak etal (Lab. Invest. 57(6):673-686 (1987)). The implants were then removedand directly analyzed using an inverted microscope. The analysisinvolved counting the number of blood vessels that had grown into thefibrin in the control and experimental group.

[0050] Briefly, 4 fibrin implants were surgically implantedsubcutaneously into Fisher 344 rats (2 dorsal and 2 ventral sides). Theaverage rat weight was approximately 125 grams.

[0051] Three rats (EXP) were given tail vein injections of eithercontrol (fibrin alone), 100 μl of 100 μg/ml of 7S domain of type IVcollagen (approximately 0.80 mg/kg body weight), 100 μl of 100 μg/ml oftype IV collagen hexamer (approximately 0.80 mg/kg body weight), orrecombinant collagen type IV NCl (α3) monomer at a concentration of 1.26mg/ml in PBS (120 μg protein, or approximately 0.96 mg/kg body weight)and 3 rats (C) were given 100 μl tail vein injections of PBS. Injectionsof recombinant protein were given every other day for five doses. Theinjection schedule was as follows:

[0052] Day 1: (implant day) injection and remove blood sample (EXP andC)

[0053] Day 3: Injection (EXP and C)

[0054] Day 5: Injection and remove blood sample (EXP and C)

[0055] Day 7: Injection (EXP and C)

[0056] Day 9: Injection and remove blood sample (EXP and C)

[0057] Day 1 1: Remove and fix implants (save blood sample) (EXP and C)

[0058] The results of one experiment were as follows: 2 week in vivoexperiment: Control (fibrin alone) about 66 BV 7S domain of type IV lenscollagen (100 μg/ml) None Hexamer of type IV lens collagen (100 μg/ml)None Monomer (α3) None

[0059] The results are shown as the mean number of blood vessels perimplant. The results of this study demonstrate that isolated domains oftype IV collagen, including the α3 monomer, can significantly inhibitcapillary growth in the in vivo fibrin clot implant model. In subsequentexperiments, the inhibitory effect was occasionally seen to attenuatewith time, suggesting that higher dosages or more frequent injectionsmight be even more effective.

[0060] A similar experiment was conducted using recombinant human typeIV collagen NCl (α1) monomer (100 μl of a 1 μg/μl solution;approximately 0.80 mg/kg body weight) and comparing the number of bloodvessels that had grown into the fibrin at day 11 of treatment relativeto the control group. Three rats per group were analyzed with each rathaving 4 implants. These experiments demonstrated that administration ofthe α1 monomer significantly inhibited capillary growth in the in vivofibrin clot implant model (FIG. 5).

EXAMPLE 3 Recombinant NCl (α2) Domain Inhibits Angiogenesis In Vivo

[0061] We next tested the effects of systemic administration of solubleNCl α-chain monomers in the chick embryo CAM angiogenesis assay.

[0062] Angiogenesis was induced in the CAMs of 10 day old chick embryoswith bFGF as described (Brooks et al., Cell 92:391-400 (1998)). Twentyfour hours later the embryos were systemically treated with variousconcentrations of recombinant NCl α-chain monomers, in a total volume of100 μl of sterile phosphate buffered saline (PBS). Two days later theembryos were sacrificed and the filter discs and CAM tissues removed.Angiogenesis was quantitated by counting the number of angigogenic bloodvessel branch points in the confined area of the filter disc. TheAngiogenic Index is defined as the number of branch points fromexperimental treatment minus control treatment.

[0063] In initial experiments, recombinant α1 or α2 NCl domains wereinjected at a concentration of 50 μg per embryo. At this concentration,the NCl domains were shown to be highly toxic as demonstrated by greaterthan 90% embryo cell death. However, at lower doses they were welltolerated and showed potent anti-angiogenic activity. A total of 6individual angiogenesis experiments were conducted with the NCl domains.However, in two experiments, the bFGF induction was low, making itdifficult to interpret the results. The NCl α2 domain appeared to bemore consistent and potent than the α1 NCl domain at inhibitingangiogenesis. In fact, systemic administration of 30 μg of NCl α2consistently inhibited angiogenesis by greater than 90% (FIGS. 6-9), asmeasured by inhibition of the bFGF-induced increase in the angiogenicindex and the mean number of blood vessel branch points. In contrast,NCl α1 domain showed variable inhibitory activity (0%-50%) throughoutthe experiments.

EXAMPLE 4 Recombinant NCl Domain Inhibits Melanoma Tumor Growth In Vivo

[0064] Since the growth of all solid tumors depends on angiogenesis toprovide nutrients for its continued expansion, reagents that have thecapacity to inhibit angiogenesis may significantly inhibit tumor growth.Therefore, we tested the effects of recombinant NCl domains of type IVcollagen for their effects on tumor growth in vivo.

[0065] To test the effects of NCl domains on tumor growth in vivo, weutilized the chick embryo tumor growth assay. Briefly, single cellsuspensions of 3 distinct tumor types were applied to the CAM of 10 dayold chick embryos. The tumors included CS-1 Melanoma cells (5×10⁶),HT1080 human fibrosarcoma cells (4×10⁵) and Hep-3 human epidermoidcarcinoma cells (2×10⁵). The embryos were injected systemically withvarying concentrations of NCl α-chain monomers 24 hours later. Theembryos were next allowed to incubate for a total of 7 days, at whichtime they were sacrificed. The resulting tumors were resected and wetweights determined. A total of 6 tumor growth assays were conducted withthe 3 distinct tumor types. A single injection of 10 μg NC1 α2 domaininhibited CS1 melanoma tumor growth by approximately 70% relative tocontrol (FIG. 10). In similar experiments, dose response curves werecompleted with CS-1 tumors. Systemic administration of NC1 α2 resultedin a dose-dependent inhibition of CS-1 melanoma tumor growth in vivowith a maximum inhibition following a single dose at 30 μg (FIG. 11).Systemic administration of NCl α1 also inhibited CS-1 tumor growth butit was variable and in some experiments failed to inhibit tumor growth(See FIG. 10). In similar experiments, NCl α2 inhibited HT1080 humanfibrosarcoma tumor growth by approximately 50% after a single systemicinjection of 30 μg, while NCl α1 and α4 had no effect (FIG. 12).Finally, systemic administration of NCl α2 (30.0 μg) and α3 inhibitedHep-3 human epidermoid carcinoma tumor growth by approximately 40% and60% respectively, and al inhibited Hep-3 tumor growth by approximately30%, while NCl α5 domain failed to inhibit tumor growth (FIG. 13).

[0066] We conclude from these in vivo studies that tumor growth can beinhibited by isolated NCl α-chain monomers. These molecules can thus beused alone, or to complement the use of existing anti-tumor agents, inproviding enhanced and more effective anti-tumor therapy.

EXAMPLE 5 Immobilized NCl Domains Support Human Endothelial CellAdhesion

[0067] In order for new blood vessels to form, endothelial cells musthave the capacity to adhere and migrate through the ECM. Moreover, thisendothelial cell-ECM interaction may facilitate signal transductionevents required for new blood vessel formation. Therefore, since typeIV-collagen is an ECM protein which is known to support cell adhesion,we tested the ability of the NCl domains to support endothelial cellattachment.

[0068] Microtiter plates were coated with 25 μg/ml of purified NCldomains followed by incubation with 1% bovine serum albumin (BSA) toblock non-specific interactions. Human endothelial cells (ECV304) werethen allowed to attach to the immobilized NCl domains for 1 hour.Non-adherent cells were removed by washing and attached cells werequantified by measuring the optical density (O.D.) of crystal violeteluted from attached cells. Data bars represent the mean+/− standarderror of the O.D. from triplicate wells.

[0069] Immobilized NCl α2, α3, and α6 domains supported endothelial celladhesion while NCl α1, α4, and α5 domains promoted little if any celladhesion (FIG. 14). Soluble NCl α1 (α1) and α2 (α2) inhibitedendothelial cell adhesion to pepsinized collagen type IV byapproximately 50% (FIG. 15).

[0070] Taken together, these findings demonstrate that isolated,recombinant NCl domains from the α1, α2, α3, and α6 chains of collagentype IV can mediate human endothelial cell adhesion and/or inhibitendothelial cell adhesion to ECM proteins in vitro, and suggest that thepotent anti-angiogenic and anti-tumor activity of the isolated NCldomains is due to disruption of endothelial cell interaction with theextracellular matrix that are necessary for angiogenesis.

EXAMPLE 6 Endothelial Cell Migration

[0071] Invasive cellular processes such as angiogenesis and tumormetastasis also require cellular motility. Thus we evaluated the abilityof isolated NCl domains to support human endothelial cell migration invitro. These experiments were conducted essentially according to themethods in Brooks et al., J. Clin. Invest. 99:1390-1398 (1997).

[0072] The results of these experiments indicate that NCl α2, α3, and α6domains can support human endothelial cell migration in vitro, while α1,α4, and α5 domains showed little if any capacity to support endothelialcell migration (FIG. 16).

EXAMPLE 7 Efficacy in Lewis Lung In Vivo Tumor

[0073] The above studies indicated that specific domains of collagentype IV can promote cell migration in vitro. Thus, we evaluated theability of NCl domains to support endothelial cell migration in vivo.

[0074] The α (IV) NCl domain hexamer, isolated by enzymatic digestion ofbovine lens capsule basement membrane by known protocols (Peczon et al.,Exp. Eye Res. 30:155-165 (1980)) was tested in the metastatic Lewis lungmouse tumor model using a standard protocol which is considered to be agood model of both metastasis and angiogenesis of lung tumors. (See forexample, Teicher et al., Anticancer Res. 18:2567-2573 (1998); Guibaud etal., Anticancer Drugs 8:276-282 (1997); Anderson et al., Cancer Res.56:715-718 (1996)).

[0075] Each study consisted of an untreated control group and sixtreatment groups. There were ten animals per treatment group with 40mice in the control. In each study, all treatment was administeredintravenously once every 2 days for 7 doses starting one day after tumorinoculation. Dosages of α (IV) NCl hexamer were either 100 μg/mouse or200 μg/mouse. In the Lewis lung study, the tumor cell inoculum was 1×10⁶viable cells. All animals were weighed twice a week throughout thestudy. Starting one day after the last treatment, 5 mice wereperiodically sacrificed from each control group to measure pulmonarytumor burden. The experiment was terminated at day 14 when the lungs ofthe control animals had sufficient tumor mass to provide meaningfulevaluation. At that time, the lungs of all remaining animals wereexcised, weighed, and the number of tumor foci greater than 2 mm indiameter counted. The resulting data showed that both dosages of a (IV)NCl hexamer significantly reduced the number of visible lung metastases(Mann-Whitney Rank Sum Test, p<0.05), with 8 visible lung metastases inthe control, vs. 5 (100 μg/mouse) and 4 (200 jig/mouse), and the 100μg/mouse dosage reduced the lung weights from a median of 520 mg incontrols to a median of 462 mg in experimental, while the median lungweight of mice treated with 200 μg/mouse was 620 mg.

[0076] Other in vivo studies demonstrated that tumor cell metastasis tothe lung can be reduced by 50% or more using intravenous injections ofthe Type IV collagen domains in murine B16 melanoma, human A375SMmelanoma xenografts. Furthermore, injection of the NCl hexamer alsosignificantly reduced the number of lung tumors in separate Lewis Lungtumor studies.

[0077] We conclude from all of the above studies that angiogenesis,tumor growth and metastasis, and endothelial cell adhesion to the ECM,can be inhibited by isolated, recombinant domains of type IV collagen.The present invention is thus broadly applicable to a variety of useswhich include inhibition of angiogenesis and treatment of diseases andconditions with accompanying undesired angiogenesis, such as solid andblood-borne tumors including but not limited to melanomas, carcinomas,sarcomas, rhabdomyosarcoma, retinoblastoma., Ewing sarcoma,neuroblastoma, osteosarcoma, and leukemia.

[0078] The invention is further applicable to treating non-tumorigenicdiseases and conditions with accompanying undesired angiogenesis,including but not limited to diabetic retinopathy, rheumatoid arthritis,retinal neovascularization, choroidal neovascularization, maculardegeneration., corneal neovascularization, retinopathy of prematurity.,corneal graft rejection, neovascular glaucoma., retrolental fibroplasia,epidemic keratoconjunctivitis, Vitamin A deficiency, contact lensoverwear, atopic keratitis, superior limbic keratitis, pterygiumkeratitis sicca, sogrens, acne rosacea, phylectenulosis, syphilis,Mycobacteria infections, lipid degeneration, chemical bums, bacterialulcers, fungal ulcers, Herpes simplex infections, Herpes zosterinfections, protozoan infections, Kaposi's sarcoma, Mooren ulcer,Terrien's marginal degeneration, marginal keratolysis, traum, systemiclupus, polyarteritis, Wegeners sarcoidosis, scleritis, Steven's Johnsondisease, radial keratotomy, sickle cell anemia, sarcoid, pseudoxanthomaelasticum, Pagets disease, vein occlusion, artery occulsion, carotidobstructive disease, chronic uveitis, chronic vitritis, Lyme's disease,Eales disease, Bechets disease, myopia, optic pits, Stargarts disease,pars planitis, chronic retinal detachment, hyperviscosity syndromes,toxoplasmosis, post-laser complications, abnormal proliferation offibrovascular tissue, hemangiomas, Osler-Weber-Rendu, acquired immunedeficiency syndrome, ocular neovascular disease, osteoarthritis, chronicinflammation, Crohn's disease, ulceritive colitis, psoriasis.,atherosclerosis, and pemphigoid. See U.S. Pat. No. 5,712,291)

[0079] The invention is also broadly applicable to methods forinhibiting tumor growth and metastasis, reduction of scar tissueformation, reduction of complications due to cell adhesion in organtransplants, and the inhibition of lymphocyte adhesion and mobility.

[0080] While the fundamental novel features of the invention have beenshown and described, it will be understood that various omissions,substitutions, and changes in the form and details illustrated may bemade by those skilled in the art without departing from the spirit ofthe invention. For example, various modifications, additions, and/orsubstitutions can be made to the type IV collagen α monomer chains thatwould be encompassed by the invention. It is the intention, therefore,to be limited only as indicated by the scope of the following claims:

1 12 1 900 DNA Human CDS (40)..(819) 1 ctgccgcctg cctgcctgcc actgagggttcccagcacc atg agg gcc tgg atc 54 Met Arg Ala Trp Ile 1 5 ttc ttt ctc ctttgc ctg gcc ggg agg gct ctg gca gcc cca cta gcc 102 Phe Phe Leu Leu CysLeu Ala Gly Arg Ala Leu Ala Ala Pro Leu Ala 10 15 20 gac tac aag gac gacgat gac aag cta gca tct gtt gat cac ggc ttc 150 Asp Tyr Lys Asp Asp AspAsp Lys Leu Ala Ser Val Asp His Gly Phe 25 30 35 ctt gtg acc agg cat agtcaa aca ata gat gac cca cag tgt cct tct 198 Leu Val Thr Arg His Ser GlnThr Ile Asp Asp Pro Gln Cys Pro Ser 40 45 50 ggg acc aaa att ctt tac cacggg tac tct ttg ctc tac gtg caa ggc 246 Gly Thr Lys Ile Leu Tyr His GlyTyr Ser Leu Leu Tyr Val Gln Gly 55 60 65 aat gaa cgg gcc cat ggc cag gacttg ggc acg gcc ggc agc tgc ctg 294 Asn Glu Arg Ala His Gly Gln Asp LeuGly Thr Ala Gly Ser Cys Leu 70 75 80 85 cgc aag ttc agc aca atg ccc ttcctg ttc tgc aat att aac aac gtg 342 Arg Lys Phe Ser Thr Met Pro Phe LeuPhe Cys Asn Ile Asn Asn Val 90 95 100 tgc aac ttt gca tca cga aat gactac tcg tac tgg ctg tcc acc cct 390 Cys Asn Phe Ala Ser Arg Asn Asp TyrSer Tyr Trp Leu Ser Thr Pro 105 110 115 gag ccc atg ccc atg tca atg gcaccc atc acg ggg gaa aac ata aga 438 Glu Pro Met Pro Met Ser Met Ala ProIle Thr Gly Glu Asn Ile Arg 120 125 130 cca ttt att agt agg tgt gct gtgtgt gag gcg cct gcc atg gtg atg 486 Pro Phe Ile Ser Arg Cys Ala Val CysGlu Ala Pro Ala Met Val Met 135 140 145 gcc gtg cac agc cag acc att cagatc cca ccg tgc ccc agc ggg tgg 534 Ala Val His Ser Gln Thr Ile Gln IlePro Pro Cys Pro Ser Gly Trp 150 155 160 165 tcc tcg ctg tgg atc ggc tactct ttt gtg atg cac acc agc gct ggt 582 Ser Ser Leu Trp Ile Gly Tyr SerPhe Val Met His Thr Ser Ala Gly 170 175 180 gca gaa ggc tct ggc caa gccctg gcg tcc ccc ggc tcc tgc ctg gag 630 Ala Glu Gly Ser Gly Gln Ala LeuAla Ser Pro Gly Ser Cys Leu Glu 185 190 195 gag ttt aga agt gcg cca ttcatc gag tgt cac ggc cgt ggg acc tgc 678 Glu Phe Arg Ser Ala Pro Phe IleGlu Cys His Gly Arg Gly Thr Cys 200 205 210 aat tac tac gca aac gct tacagc ttt tgg ctc gcc acc ata gag agg 726 Asn Tyr Tyr Ala Asn Ala Tyr SerPhe Trp Leu Ala Thr Ile Glu Arg 215 220 225 agc gag atg ttc aag aag cctacg ccg tcc acc ttg aag gca ggg gag 774 Ser Glu Met Phe Lys Lys Pro ThrPro Ser Thr Leu Lys Ala Gly Glu 230 235 240 245 ctg cgc acg cac gtc agccgc tgc caa gtc tgt atg aga aga aca 819 Leu Arg Thr His Val Ser Arg CysGln Val Cys Met Arg Arg Thr 250 255 260 taatgaagcc tgactcagct accgcgggccctattctata gtgtcaccta aatgctagag 879 ctcgctgatc agcctcgact g 900 2 260PRT Human 2 Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg AlaLeu 1 5 10 15 Ala Ala Pro Leu Ala Asp Tyr Lys Asp Asp Asp Asp Lys LeuAla Ser 20 25 30 Val Asp His Gly Phe Leu Val Thr Arg His Ser Gln Thr IleAsp Asp 35 40 45 Pro Gln Cys Pro Ser Gly Thr Lys Ile Leu Tyr His Gly TyrSer Leu 50 55 60 Leu Tyr Val Gln Gly Asn Glu Arg Ala His Gly Gln Asp LeuGly Thr 65 70 75 80 Ala Gly Ser Cys Leu Arg Lys Phe Ser Thr Met Pro PheLeu Phe Cys 85 90 95 Asn Ile Asn Asn Val Cys Asn Phe Ala Ser Arg Asn AspTyr Ser Tyr 100 105 110 Trp Leu Ser Thr Pro Glu Pro Met Pro Met Ser MetAla Pro Ile Thr 115 120 125 Gly Glu Asn Ile Arg Pro Phe Ile Ser Arg CysAla Val Cys Glu Ala 130 135 140 Pro Ala Met Val Met Ala Val His Ser GlnThr Ile Gln Ile Pro Pro 145 150 155 160 Cys Pro Ser Gly Trp Ser Ser LeuTrp Ile Gly Tyr Ser Phe Val Met 165 170 175 His Thr Ser Ala Gly Ala GluGly Ser Gly Gln Ala Leu Ala Ser Pro 180 185 190 Gly Ser Cys Leu Glu GluPhe Arg Ser Ala Pro Phe Ile Glu Cys His 195 200 205 Gly Arg Gly Thr CysAsn Tyr Tyr Ala Asn Ala Tyr Ser Phe Trp Leu 210 215 220 Ala Thr Ile GluArg Ser Glu Met Phe Lys Lys Pro Thr Pro Ser Thr 225 230 235 240 Leu LysAla Gly Glu Leu Arg Thr His Val Ser Arg Cys Gln Val Cys 245 250 255 MetArg Arg Thr 260 3 900 DNA Human CDS (40)..(813) 3 ctgccgcctg cctgcctgccactgagggtt cccagcacc atg agg gcc tgg atc 54 Met Arg Ala Trp Ile 1 5 ttcttt ctc ctt tgc ctg gcc ggg agg gct ctg gca gcc cca cta gcc 102 Phe PheLeu Leu Cys Leu Ala Gly Arg Ala Leu Ala Ala Pro Leu Ala 10 15 20 gac tacaag gac gac gat gac aag cta gcc gtc agc atc ggc tac ctc 150 Asp Tyr LysAsp Asp Asp Asp Lys Leu Ala Val Ser Ile Gly Tyr Leu 25 30 35 ctg gtg aagcac agc cag acg gac cag gag ccc atg tgc ccg gtg ggc 198 Leu Val Lys HisSer Gln Thr Asp Gln Glu Pro Met Cys Pro Val Gly 40 45 50 atg aac aaa ctctgg agt gga tac agc ctg ctg tac ttc gag ggc cag 246 Met Asn Lys Leu TrpSer Gly Tyr Ser Leu Leu Tyr Phe Glu Gly Gln 55 60 65 gag aag gcg cac aaccag gac ctg ggg ctg gcg ggc tcc tgc ctg gcg 294 Glu Lys Ala His Asn GlnAsp Leu Gly Leu Ala Gly Ser Cys Leu Ala 70 75 80 85 cgg ttc agc acc atgccc ttc ctg tac tgc aac cct ggt gat gtc tgc 342 Arg Phe Ser Thr Met ProPhe Leu Tyr Cys Asn Pro Gly Asp Val Cys 90 95 100 tac tat gcc agc cggaac gac aag tcc tac tgg ctc tct acc act gcg 390 Tyr Tyr Ala Ser Arg AsnAsp Lys Ser Tyr Trp Leu Ser Thr Thr Ala 105 110 115 ccg ctg ccc atg atgccc gtg gcc gag gac gag atc aag ccc tac atc 438 Pro Leu Pro Met Met ProVal Ala Glu Asp Glu Ile Lys Pro Tyr Ile 120 125 130 agc cgc tgt tct gtgtgt gag gcc ccg gcc atc gcc atc gcg gtc cac 486 Ser Arg Cys Ser Val CysGlu Ala Pro Ala Ile Ala Ile Ala Val His 135 140 145 agt cag gat gtc tccatc cca cac tgc cca gct ggg tgg cgg agt ttg 534 Ser Gln Asp Val Ser IlePro His Cys Pro Ala Gly Trp Arg Ser Leu 150 155 160 165 tgg atc gga tattcc ttc ctc atg cac acg gcg gcg gga gac gaa ggc 582 Trp Ile Gly Tyr SerPhe Leu Met His Thr Ala Ala Gly Asp Glu Gly 170 175 180 ggt ggc caa tcactg gtg tca ccg ggc agc tgt cta gag gac ttc cgc 630 Gly Gly Gln Ser LeuVal Ser Pro Gly Ser Cys Leu Glu Asp Phe Arg 185 190 195 gcc aca cca ttcatc gaa tgc aat gga ggc cgc ggc acc tgc cac tac 678 Ala Thr Pro Phe IleGlu Cys Asn Gly Gly Arg Gly Thr Cys His Tyr 200 205 210 tac gcc aac aagtac agc ttc tgg ctg acc acc att ccc gag cag agc 726 Tyr Ala Asn Lys TyrSer Phe Trp Leu Thr Thr Ile Pro Glu Gln Ser 215 220 225 ttc cag ggc tcgccc tcc gcc gac acg ctc aag gcc ggc ctc atc cgc 774 Phe Gln Gly Ser ProSer Ala Asp Thr Leu Lys Ala Gly Leu Ile Arg 230 235 240 245 aca cac atcagc cgc tgc cag gtg tgc atg aag aac ctg tgagccggcg 823 Thr His Ile SerArg Cys Gln Val Cys Met Lys Asn Leu 250 255 cgtgccaggg ccctattctatagtgtcacc taaatgctag agctcgctga tcagcctcga 883 ctgtgccttc tagttgc 900 4258 PRT Human 4 Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly ArgAla Leu 1 5 10 15 Ala Ala Pro Leu Ala Asp Tyr Lys Asp Asp Asp Asp LysLeu Ala Val 20 25 30 Ser Ile Gly Tyr Leu Leu Val Lys His Ser Gln Thr AspGln Glu Pro 35 40 45 Met Cys Pro Val Gly Met Asn Lys Leu Trp Ser Gly TyrSer Leu Leu 50 55 60 Tyr Phe Glu Gly Gln Glu Lys Ala His Asn Gln Asp LeuGly Leu Ala 65 70 75 80 Gly Ser Cys Leu Ala Arg Phe Ser Thr Met Pro PheLeu Tyr Cys Asn 85 90 95 Pro Gly Asp Val Cys Tyr Tyr Ala Ser Arg Asn AspLys Ser Tyr Trp 100 105 110 Leu Ser Thr Thr Ala Pro Leu Pro Met Met ProVal Ala Glu Asp Glu 115 120 125 Ile Lys Pro Tyr Ile Ser Arg Cys Ser ValCys Glu Ala Pro Ala Ile 130 135 140 Ala Ile Ala Val His Ser Gln Asp ValSer Ile Pro His Cys Pro Ala 145 150 155 160 Gly Trp Arg Ser Leu Trp IleGly Tyr Ser Phe Leu Met His Thr Ala 165 170 175 Ala Gly Asp Glu Gly GlyGly Gln Ser Leu Val Ser Pro Gly Ser Cys 180 185 190 Leu Glu Asp Phe ArgAla Thr Pro Phe Ile Glu Cys Asn Gly Gly Arg 195 200 205 Gly Thr Cys HisTyr Tyr Ala Asn Lys Tyr Ser Phe Trp Leu Thr Thr 210 215 220 Ile Pro GluGln Ser Phe Gln Gly Ser Pro Ser Ala Asp Thr Leu Lys 225 230 235 240 AlaGly Leu Ile Arg Thr His Ile Ser Arg Cys Gln Val Cys Met Lys 245 250 255Asn Leu 5 900 DNA Human CDS (40)..(843) 5 ctgccgcctg cctgcctgccactgagggtt cccagcacc atg agg gcc tgg atc 54 Met Arg Ala Trp Ile 1 5 ttcttt ctc ctt tgc ctg gcc ggg agg gct ctg gca gcc ccg cta gcc 102 Phe PheLeu Leu Cys Leu Ala Gly Arg Ala Leu Ala Ala Pro Leu Ala 10 15 20 gac tacaag gac gac gat gac aaa cgt gga gac agt gga tca cct gca 150 Asp Tyr LysAsp Asp Asp Asp Lys Arg Gly Asp Ser Gly Ser Pro Ala 25 30 35 acc tgg acaacg aga ggc ttt gtc ttc acc cga cac agt caa acc aca 198 Thr Trp Thr ThrArg Gly Phe Val Phe Thr Arg His Ser Gln Thr Thr 40 45 50 gca att cct tcatgt cca gag ggg aca gtg cca ctc tac agt ggg ttt 246 Ala Ile Pro Ser CysPro Glu Gly Thr Val Pro Leu Tyr Ser Gly Phe 55 60 65 tct ttt ctt ttt gtacaa gga aat caa cga gcc cac gga caa gac ctt 294 Ser Phe Leu Phe Val GlnGly Asn Gln Arg Ala His Gly Gln Asp Leu 70 75 80 85 gga act ctt ggc agctgc ctg cag cga ttt acc aca atg cca ttc tta 342 Gly Thr Leu Gly Ser CysLeu Gln Arg Phe Thr Thr Met Pro Phe Leu 90 95 100 ttc tgc aat gtc aatgat gta tgt aat ttt gca tct cga aat gat tat 390 Phe Cys Asn Val Asn AspVal Cys Asn Phe Ala Ser Arg Asn Asp Tyr 105 110 115 tca tac tgg ctg tcaaca cca gct ctg atg cca atg aac atg gct ccc 438 Ser Tyr Trp Leu Ser ThrPro Ala Leu Met Pro Met Asn Met Ala Pro 120 125 130 att act ggc aga gccctt gag cct tat ata agc aga tgc act gtt tgt 486 Ile Thr Gly Arg Ala LeuGlu Pro Tyr Ile Ser Arg Cys Thr Val Cys 135 140 145 gaa ggt cct gcg atcgcc ata gcc gtt cac agc caa acc act gac att 534 Glu Gly Pro Ala Ile AlaIle Ala Val His Ser Gln Thr Thr Asp Ile 150 155 160 165 cct cca tgt cctcac ggc tgg att tct ctc tgg aaa gga ttt tca ttc 582 Pro Pro Cys Pro HisGly Trp Ile Ser Leu Trp Lys Gly Phe Ser Phe 170 175 180 atc atg ttc acaagt gca ggt tct gag ggc gcc ggg caa gca ctg gcc 630 Ile Met Phe Thr SerAla Gly Ser Glu Gly Ala Gly Gln Ala Leu Ala 185 190 195 tcc ccc ggc tcctgc ctg gaa gaa ttc cga gcc agc cca ttt cta gaa 678 Ser Pro Gly Ser CysLeu Glu Glu Phe Arg Ala Ser Pro Phe Leu Glu 200 205 210 tgt cat gga agagga acg tgc aac tac tat tca aat tcc tac agt ttc 726 Cys His Gly Arg GlyThr Cys Asn Tyr Tyr Ser Asn Ser Tyr Ser Phe 215 220 225 tgg ctg gct tcatta aac cca gaa aga atg ttc aga aag cct att cca 774 Trp Leu Ala Ser LeuAsn Pro Glu Arg Met Phe Arg Lys Pro Ile Pro 230 235 240 245 tca act gtgaaa gct ggg gaa tta gaa aaa ata ata agt cgc tgt cag 822 Ser Thr Val LysAla Gly Glu Leu Glu Lys Ile Ile Ser Arg Cys Gln 250 255 260 gtg tgc atgaag aaa aga cac tgagggccct attctatagt gtcacctaaa 873 Val Cys Met Lys LysArg His 265 tgctagagct cgctgatcag cctcgac 900 6 268 PRT Human 6 Met ArgAla Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu 1 5 10 15 AlaAla Pro Leu Ala Asp Tyr Lys Asp Asp Asp Asp Lys Arg Gly Asp 20 25 30 SerGly Ser Pro Ala Thr Trp Thr Thr Arg Gly Phe Val Phe Thr Arg 35 40 45 HisSer Gln Thr Thr Ala Ile Pro Ser Cys Pro Glu Gly Thr Val Pro 50 55 60 LeuTyr Ser Gly Phe Ser Phe Leu Phe Val Gln Gly Asn Gln Arg Ala 65 70 75 80His Gly Gln Asp Leu Gly Thr Leu Gly Ser Cys Leu Gln Arg Phe Thr 85 90 95Thr Met Pro Phe Leu Phe Cys Asn Val Asn Asp Val Cys Asn Phe Ala 100 105110 Ser Arg Asn Asp Tyr Ser Tyr Trp Leu Ser Thr Pro Ala Leu Met Pro 115120 125 Met Asn Met Ala Pro Ile Thr Gly Arg Ala Leu Glu Pro Tyr Ile Ser130 135 140 Arg Cys Thr Val Cys Glu Gly Pro Ala Ile Ala Ile Ala Val HisSer 145 150 155 160 Gln Thr Thr Asp Ile Pro Pro Cys Pro His Gly Trp IleSer Leu Trp 165 170 175 Lys Gly Phe Ser Phe Ile Met Phe Thr Ser Ala GlySer Glu Gly Ala 180 185 190 Gly Gln Ala Leu Ala Ser Pro Gly Ser Cys LeuGlu Glu Phe Arg Ala 195 200 205 Ser Pro Phe Leu Glu Cys His Gly Arg GlyThr Cys Asn Tyr Tyr Ser 210 215 220 Asn Ser Tyr Ser Phe Trp Leu Ala SerLeu Asn Pro Glu Arg Met Phe 225 230 235 240 Arg Lys Pro Ile Pro Ser ThrVal Lys Ala Gly Glu Leu Glu Lys Ile 245 250 255 Ile Ser Arg Cys Gln ValCys Met Lys Lys Arg His 260 265 7 900 DNA Human CDS (40)..(819) 7ctgccgcctg cctgcctgcc actgagggtt cccagcacc atg agg gcc tgg atc 54 MetArg Ala Trp Ile 1 5 ttc ttt ctc ctt tgc ctg gcc ggg agg gct ctg gca gccccg cta gcc 102 Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu Ala Ala ProLeu Ala 10 15 20 gac tac aag gac gac gat gac aag cct gga tac ctc ggt ggcttc ctc 150 Asp Tyr Lys Asp Asp Asp Asp Lys Pro Gly Tyr Leu Gly Gly PheLeu 25 30 35 ctg gtt ctc cac agt cag acg gac cag gag ccc acc tgc ccc ctgggc 198 Leu Val Leu His Ser Gln Thr Asp Gln Glu Pro Thr Cys Pro Leu Gly40 45 50 atg ccc agg ctc tgg act ggg tat agt ctg tta tac ctg gaa ggg caa246 Met Pro Arg Leu Trp Thr Gly Tyr Ser Leu Leu Tyr Leu Glu Gly Gln 5560 65 gag aaa gct cac aat caa gac ctt ggt ctg gca ggg tct tgc ctt ccc294 Glu Lys Ala His Asn Gln Asp Leu Gly Leu Ala Gly Ser Cys Leu Pro 7075 80 85 gta ttt agc acg ctg ccc ttt gcc tac tgc aac atc cac cag gtg tgc342 Val Phe Ser Thr Leu Pro Phe Ala Tyr Cys Asn Ile His Gln Val Cys 9095 100 cac tat gcc cag aga aac gac aga tcc tac tgg ctg gcc agc gct gcg390 His Tyr Ala Gln Arg Asn Asp Arg Ser Tyr Trp Leu Ala Ser Ala Ala 105110 115 ccc ctc ccc atg atg cca ctc tct gaa gag gcg atc cgc ccc tat gtc438 Pro Leu Pro Met Met Pro Leu Ser Glu Glu Ala Ile Arg Pro Tyr Val 120125 130 agc cgc tgt gcg gta tgc gag gcc ccg gcc cag gcg gtg gcg gtg cac486 Ser Arg Cys Ala Val Cys Glu Ala Pro Ala Gln Ala Val Ala Val His 135140 145 agc cag gac cag tcc atc ccc cca tgt ccg cag acc tgg agg agc ctc534 Ser Gln Asp Gln Ser Ile Pro Pro Cys Pro Gln Thr Trp Arg Ser Leu 150155 160 165 tgg atc ggg tat tca ttc ctg atg cac aca gga gct ggg gac caagga 582 Trp Ile Gly Tyr Ser Phe Leu Met His Thr Gly Ala Gly Asp Gln Gly170 175 180 gga ggg cag gcc ctt atg tca cct ggc agc tgc ctg gaa gat ttcaga 630 Gly Gly Gln Ala Leu Met Ser Pro Gly Ser Cys Leu Glu Asp Phe Arg185 190 195 gca gca cca ttc ctt gaa tgc cag ggc cgg cag gga act tgc cacttt 678 Ala Ala Pro Phe Leu Glu Cys Gln Gly Arg Gln Gly Thr Cys His Phe200 205 210 ttc gca aat aag tat agc ttc tgg ctc aca acg gtg aaa gca gacttg 726 Phe Ala Asn Lys Tyr Ser Phe Trp Leu Thr Thr Val Lys Ala Asp Leu215 220 225 cag ttt tcc tct gct cca gca cca gac acc tta aaa gaa agc caggcc 774 Gln Phe Ser Ser Ala Pro Ala Pro Asp Thr Leu Lys Glu Ser Gln Ala230 235 240 245 caa cgc cag aaa atc agc cgg tgc cag gtc tgc gtg aag tatagc 819 Gln Arg Gln Lys Ile Ser Arg Cys Gln Val Cys Val Lys Tyr Ser 250255 260 taggggccct attctatagt gtcacctaaa tgctagagct cgctgatcagcctcgactgt 879 gccttctagt tgccagccat c 900 8 260 PRT Human 8 Met Arg AlaTrp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu 1 5 10 15 Ala AlaPro Leu Ala Asp Tyr Lys Asp Asp Asp Asp Lys Pro Gly Tyr 20 25 30 Leu GlyGly Phe Leu Leu Val Leu His Ser Gln Thr Asp Gln Glu Pro 35 40 45 Thr CysPro Leu Gly Met Pro Arg Leu Trp Thr Gly Tyr Ser Leu Leu 50 55 60 Tyr LeuGlu Gly Gln Glu Lys Ala His Asn Gln Asp Leu Gly Leu Ala 65 70 75 80 GlySer Cys Leu Pro Val Phe Ser Thr Leu Pro Phe Ala Tyr Cys Asn 85 90 95 IleHis Gln Val Cys His Tyr Ala Gln Arg Asn Asp Arg Ser Tyr Trp 100 105 110Leu Ala Ser Ala Ala Pro Leu Pro Met Met Pro Leu Ser Glu Glu Ala 115 120125 Ile Arg Pro Tyr Val Ser Arg Cys Ala Val Cys Glu Ala Pro Ala Gln 130135 140 Ala Val Ala Val His Ser Gln Asp Gln Ser Ile Pro Pro Cys Pro Gln145 150 155 160 Thr Trp Arg Ser Leu Trp Ile Gly Tyr Ser Phe Leu Met HisThr Gly 165 170 175 Ala Gly Asp Gln Gly Gly Gly Gln Ala Leu Met Ser ProGly Ser Cys 180 185 190 Leu Glu Asp Phe Arg Ala Ala Pro Phe Leu Glu CysGln Gly Arg Gln 195 200 205 Gly Thr Cys His Phe Phe Ala Asn Lys Tyr SerPhe Trp Leu Thr Thr 210 215 220 Val Lys Ala Asp Leu Gln Phe Ser Ser AlaPro Ala Pro Asp Thr Leu 225 230 235 240 Lys Glu Ser Gln Ala Gln Arg GlnLys Ile Ser Arg Cys Gln Val Cys 245 250 255 Val Lys Tyr Ser 260 9 900DNA Human CDS (40)..(831) 9 ctgccgcctg cctgcctgcc actgagggtt cccagcaccatg agg gcc tgg atc 54 Met Arg Ala Trp Ile 1 5 ttc ttt ctc ctt tgc ctggcc ggg agg gct ctg gca gcc ccg cta gct 102 Phe Phe Leu Leu Cys Leu AlaGly Arg Ala Leu Ala Ala Pro Leu Ala 10 15 20 gac tac aag gac gac gat gacaaa ggt ccc cct gga acc tcc tct gtt 150 Asp Tyr Lys Asp Asp Asp Asp LysGly Pro Pro Gly Thr Ser Ser Val 25 30 35 gca cat gga ttt ctt att aca cgccac agc cag aca acg gat gca cca 198 Ala His Gly Phe Leu Ile Thr Arg HisSer Gln Thr Thr Asp Ala Pro 40 45 50 caa tgc cca cag gga aca ctt cag gtctat gaa ggc ttt tct ctc ctg 246 Gln Cys Pro Gln Gly Thr Leu Gln Val TyrGlu Gly Phe Ser Leu Leu 55 60 65 tat gta caa gga aat aaa aga gcc cac ggtcaa gac ttg ggg acg gct 294 Tyr Val Gln Gly Asn Lys Arg Ala His Gly GlnAsp Leu Gly Thr Ala 70 75 80 85 ggc agc tgc ctt cgt cgc ttt agt acc atgcct ttc atg ttc tgc aac 342 Gly Ser Cys Leu Arg Arg Phe Ser Thr Met ProPhe Met Phe Cys Asn 90 95 100 atc aat aat gtt tgc aac ttt gct tca agaaat gac tat tct tac tgg 390 Ile Asn Asn Val Cys Asn Phe Ala Ser Arg AsnAsp Tyr Ser Tyr Trp 105 110 115 ctc tct acc cca gag ccc atg cca atg agcatg caa ccc cta aag ggc 438 Leu Ser Thr Pro Glu Pro Met Pro Met Ser MetGln Pro Leu Lys Gly 120 125 130 cag agc atc cag cca ttc att agt cga tgtgca gta tgt gaa gct cca 486 Gln Ser Ile Gln Pro Phe Ile Ser Arg Cys AlaVal Cys Glu Ala Pro 135 140 145 gct gtg gtg atc gca gtt cac agt cag acgatc cag att ccc cat tgt 534 Ala Val Val Ile Ala Val His Ser Gln Thr IleGln Ile Pro His Cys 150 155 160 165 cct cag gga tgg gat tct ctg tgg attggt tat tcc ttc atg atg cat 582 Pro Gln Gly Trp Asp Ser Leu Trp Ile GlyTyr Ser Phe Met Met His 170 175 180 aca agt gca ggg gca gaa ggc tca ggtcaa gcc cta gcc tcc cct ggt 630 Thr Ser Ala Gly Ala Glu Gly Ser Gly GlnAla Leu Ala Ser Pro Gly 185 190 195 tcc tgc ttg gaa gag ttt cgt tca gctccc ttc atc gaa tgt cat ggg 678 Ser Cys Leu Glu Glu Phe Arg Ser Ala ProPhe Ile Glu Cys His Gly 200 205 210 agg ggt acc tgt aac tac tat gcc aactcc tac agc ttt tgg ctg gca 726 Arg Gly Thr Cys Asn Tyr Tyr Ala Asn SerTyr Ser Phe Trp Leu Ala 215 220 225 act gta gat gtg tca gac atg ttc agtaaa cct cag tca gaa acg ctg 774 Thr Val Asp Val Ser Asp Met Phe Ser LysPro Gln Ser Glu Thr Leu 230 235 240 245 aaa gca gga gac ttg agg aca cgaatt agc cga tgt caa gtg tgc atg 822 Lys Ala Gly Asp Leu Arg Thr Arg IleSer Arg Cys Gln Val Cys Met 250 255 260 aag agg aca taacgcggccgctcgagcat gcatctagag ggccctattc 871 Lys Arg Thr tatagtgtca cctaaatgctagagctcgc 900 10 264 PRT Human 10 Met Arg Ala Trp Ile Phe Phe Leu LeuCys Leu Ala Gly Arg Ala Leu 1 5 10 15 Ala Ala Pro Leu Ala Asp Tyr LysAsp Asp Asp Asp Lys Gly Pro Pro 20 25 30 Gly Thr Ser Ser Val Ala His GlyPhe Leu Ile Thr Arg His Ser Gln 35 40 45 Thr Thr Asp Ala Pro Gln Cys ProGln Gly Thr Leu Gln Val Tyr Glu 50 55 60 Gly Phe Ser Leu Leu Tyr Val GlnGly Asn Lys Arg Ala His Gly Gln 65 70 75 80 Asp Leu Gly Thr Ala Gly SerCys Leu Arg Arg Phe Ser Thr Met Pro 85 90 95 Phe Met Phe Cys Asn Ile AsnAsn Val Cys Asn Phe Ala Ser Arg Asn 100 105 110 Asp Tyr Ser Tyr Trp LeuSer Thr Pro Glu Pro Met Pro Met Ser Met 115 120 125 Gln Pro Leu Lys GlyGln Ser Ile Gln Pro Phe Ile Ser Arg Cys Ala 130 135 140 Val Cys Glu AlaPro Ala Val Val Ile Ala Val His Ser Gln Thr Ile 145 150 155 160 Gln IlePro His Cys Pro Gln Gly Trp Asp Ser Leu Trp Ile Gly Tyr 165 170 175 SerPhe Met Met His Thr Ser Ala Gly Ala Glu Gly Ser Gly Gln Ala 180 185 190Leu Ala Ser Pro Gly Ser Cys Leu Glu Glu Phe Arg Ser Ala Pro Phe 195 200205 Ile Glu Cys His Gly Arg Gly Thr Cys Asn Tyr Tyr Ala Asn Ser Tyr 210215 220 Ser Phe Trp Leu Ala Thr Val Asp Val Ser Asp Met Phe Ser Lys Pro225 230 235 240 Gln Ser Glu Thr Leu Lys Ala Gly Asp Leu Arg Thr Arg IleSer Arg 245 250 255 Cys Gln Val Cys Met Lys Arg Thr 260 11 900 DNA HumanCDS (40)..(819) 11 ctgccgcctg cctgcctgcc actgagggtt cccagcacc atg agggcc tgg atc 54 Met Arg Ala Trp Ile 1 5 ttc ttt ctc ctt tgc ctg gcc gggagg gct ctg gca gcc cca cta gcc 102 Phe Phe Leu Leu Cys Leu Ala Gly ArgAla Leu Ala Ala Pro Leu Ala 10 15 20 gac tac aag gac gac gat gac aag ctagcg agc atg aga gtg ggc tac 150 Asp Tyr Lys Asp Asp Asp Asp Lys Leu AlaSer Met Arg Val Gly Tyr 25 30 35 acg ttg gta aag cac agc cag tcg gaa caggtg ccc ccg tgt ccc atc 198 Thr Leu Val Lys His Ser Gln Ser Glu Gln ValPro Pro Cys Pro Ile 40 45 50 ggg atg agc cag ctg tgg gtg ggg tac agc ttactg ttt gtg gag ggg 246 Gly Met Ser Gln Leu Trp Val Gly Tyr Ser Leu LeuPhe Val Glu Gly 55 60 65 caa gag aaa gcc cac aac cag gac ctg ggc ttt gctggc tcc tgt ctg 294 Gln Glu Lys Ala His Asn Gln Asp Leu Gly Phe Ala GlySer Cys Leu 70 75 80 85 ccc cgc ttc agc acc atg ccc ttc atc tac tgc aacatc aac gag gtg 342 Pro Arg Phe Ser Thr Met Pro Phe Ile Tyr Cys Asn IleAsn Glu Val 90 95 100 tgc cac tat gcc agg cgc aat gat aaa tct tac tggctc tcc act acc 390 Cys His Tyr Ala Arg Arg Asn Asp Lys Ser Tyr Trp LeuSer Thr Thr 105 110 115 gcc cct atc ccc atg atg ccc gtc agc cag acc cagatt ccc cag tac 438 Ala Pro Ile Pro Met Met Pro Val Ser Gln Thr Gln IlePro Gln Tyr 120 125 130 atc agc cgc tgc tct gtg tgt gag gca ccc tcg caagcc att gct gtg 486 Ile Ser Arg Cys Ser Val Cys Glu Ala Pro Ser Gln AlaIle Ala Val 135 140 145 cac agc cag gac atc acc atc ccg cag tgc ccc ctgggc tgg cgc agc 534 His Ser Gln Asp Ile Thr Ile Pro Gln Cys Pro Leu GlyTrp Arg Ser 150 155 160 165 ctc tgg att ggg tac tct ttc ctc atg cac actgcc gct ggt gcc gag 582 Leu Trp Ile Gly Tyr Ser Phe Leu Met His Thr AlaAla Gly Ala Glu 170 175 180 ggt gga ggc cag tcc ctg gtc tca cct ggc tcctgc cta gag gac ttt 630 Gly Gly Gly Gln Ser Leu Val Ser Pro Gly Ser CysLeu Glu Asp Phe 185 190 195 cgg gcc act cct ttc atc gaa tgc agt ggt gcccga ggc acc tgc cac 678 Arg Ala Thr Pro Phe Ile Glu Cys Ser Gly Ala ArgGly Thr Cys His 200 205 210 tac ttt gca aac aag tac agt ttc tgg ttg accaca gtg gag gag agg 726 Tyr Phe Ala Asn Lys Tyr Ser Phe Trp Leu Thr ThrVal Glu Glu Arg 215 220 225 cag cag ttt ggg gag ttg cct gtg tct gaa acgctg aaa gct ggg cag 774 Gln Gln Phe Gly Glu Leu Pro Val Ser Glu Thr LeuLys Ala Gly Gln 230 235 240 245 ctc cac act cga gtc agt cgc tgc cag gtgtgt atg aaa agc ctg 819 Leu His Thr Arg Val Ser Arg Cys Gln Val Cys MetLys Ser Leu 250 255 260 tagggtggca cctgccacgg gccctattct atagtgtcacctaaatgcta gagctcgctg 879 atcagcctcg actgtgcctt c 900 12 260 PRT Human12 Met Arg Ala Trp Ile Phe Phe Leu Leu Cys Leu Ala Gly Arg Ala Leu 1 510 15 Ala Ala Pro Leu Ala Asp Tyr Lys Asp Asp Asp Asp Lys Leu Ala Ser 2025 30 Met Arg Val Gly Tyr Thr Leu Val Lys His Ser Gln Ser Glu Gln Val 3540 45 Pro Pro Cys Pro Ile Gly Met Ser Gln Leu Trp Val Gly Tyr Ser Leu 5055 60 Leu Phe Val Glu Gly Gln Glu Lys Ala His Asn Gln Asp Leu Gly Phe 6570 75 80 Ala Gly Ser Cys Leu Pro Arg Phe Ser Thr Met Pro Phe Ile Tyr Cys85 90 95 Asn Ile Asn Glu Val Cys His Tyr Ala Arg Arg Asn Asp Lys Ser Tyr100 105 110 Trp Leu Ser Thr Thr Ala Pro Ile Pro Met Met Pro Val Ser GlnThr 115 120 125 Gln Ile Pro Gln Tyr Ile Ser Arg Cys Ser Val Cys Glu AlaPro Ser 130 135 140 Gln Ala Ile Ala Val His Ser Gln Asp Ile Thr Ile ProGln Cys Pro 145 150 155 160 Leu Gly Trp Arg Ser Leu Trp Ile Gly Tyr SerPhe Leu Met His Thr 165 170 175 Ala Ala Gly Ala Glu Gly Gly Gly Gln SerLeu Val Ser Pro Gly Ser 180 185 190 Cys Leu Glu Asp Phe Arg Ala Thr ProPhe Ile Glu Cys Ser Gly Ala 195 200 205 Arg Gly Thr Cys His Tyr Phe AlaAsn Lys Tyr Ser Phe Trp Leu Thr 210 215 220 Thr Val Glu Glu Arg Gln GlnPhe Gly Glu Leu Pro Val Ser Glu Thr 225 230 235 240 Leu Lys Ala Gly GlnLeu His Thr Arg Val Ser Arg Cys Gln Val Cys 245 250 255 Met Lys Ser Leu260

We claim:
 1. A method for inhibiting angiogenesis in an animal tissuecomprising contacting the tumor or animal tissue with an amounteffective to inhibit angiogenesis of a polypeptide compositioncomprising one or more isolated NCl α chain monomers of type IV collagenselected from the group consisting of α1, α2, α3, and α6 NCl chainmonomers.
 2. A method for inhibiting tumor metastasis in tissuecomprising contacting the tumor or tissue with an amount effective toinhibit tumor metastasis of a polypeptide composition comprising one ormore isolated NCl α chain monomers of type IV collagen selected from thegroup consisting of α1, α2, α3, and α6 NCl chain monomers.
 3. A methodfor inhibiting tumor growth in tissue comprising contacting the tumor ortissue with an amount effective to inhibit tumor growth of a polypeptidecomposition comprising one or more isolated NCl α chain monomers of typeIV collagen selected from the group consisting of α1, α2, α3, and α6 NClchain monomers.
 4. A kit for practicing the method of claim 1comprising: a. an amount effective to inhibit angiogenesis in an animaltissue of a polypeptide composition comprising one or more isolated NClα a chain monomers of type IV collagen selected from the groupconsisting of α1, α2, α3, and α6 NCl chain monomers, and b. instructionsfor using the amount effective of isolated NCl α chain monomers of typeIV collagen to inhibit angiogenesis in an animal tissue.
 5. A kit forpracticing the method of claim 2 comprising: a. an amount effective toinhibit tumor metastasis in tissue of an isolated domain of apolypeptide composition comprising one or more isolated NCl α chainmonomers of type IV collagen selected from the group consisting of α1,α2, α3, and α6 NCl chain monomers and b. instructions for using theamount effective of isolated NCl α chain monomers of type IV collagen toinhibit tumor metastasis in an animal tissue.
 6. A kit for practicingthe method of claim 3 comprising: a. an amount effective to inhibittumor growth in tissue of an isolated domain of a polypeptidecomposition comprising one or more isolated NCl α chain monomers of typeIV collagen selected from the group consisting of α1, α2, α3, and α6 NClchain monomers and b. instructions for using the amount effective ofisolated NCl α chain monomers of type IV collagen to inhibit tumorgrowth in an animal tissue.
 7. The method of claim 1 wherein theisolated NCl α a chain monomer of type IV collagen is the α1 chainmonomer.
 8. The method of claim 1 wherein the isolated NCl α chainmonomer of type IV collagen is the α2 chain monomer.
 9. The method ofclaim 1 wherein the isolated NCl α chain monomer of type IV collagen isthe α3 chain monomer.
 10. The method of claim 1 wherein the isolated NClα chain monomer of type IV collagen is the α6 chain monomer.
 11. Themethod of claim 2 wherein the isolated NCl α chain monomer of type IVcollagen is the α1 chain monomer.
 12. The method of claim 2 wherein theisolated NCl α chain monomer of type IV collagen is the α2 chainmonomer.
 13. The method of claim 2 wherein the isolated NCl α chainmonomer of type IV collagen is the α3 chain monomer.
 14. The method ofclaim 2 wherein the isolated NCl α chain monomer of type IV collagen isthe α6 chain monomer.
 15. The method of claim 3 wherein the isolated NClα chain monomer of type IV collagen is the α1 chain monomer.
 16. Themethod of claim 3 wherein the isolated NCl α chain monomer of type IVcollagen is the α2 chain monomer.
 17. The method of claim 3 wherein theisolated NCl α chain monomer of type IV collagen is the α3 chainmonomer.
 18. The method of claim 3 wherein the isolated NCl α chainmonomer of type IV collagen is the α6 chain monomer.
 19. A method forinhibiting endothelial cell interaction with the extracellular matrix inan animal tissue comprising contacting the tumor or animal tissue withan amount effective to inhibit endothelial cell interaction with theextracellular matrix of a polypeptide composition comprising one or moreisolated NCl α a chain monomers of type IV collagen selected from thegroup consisting of α1, α2, α3, and α6 NCl chain monomers.
 20. A kit forpracticing the method of claim 19 comprising: a. an amount effective toinhibit endothelial cell interaction with the extracellular matrix in ananimal tissue of a polypeptide composition comprising one or moreisolated NCl α chain monomers of type IV collagen selected from thegroup consisting of α1, α2, α3, and α6 NCl chain monomers, and b.instructions for using the amount effective of isolated NCl α chainmonomers of type IV collagen to inhibit endothelial cell interactionwith the extracellular matrix in an animal tissue.
 21. The method ofclaim 19 wherein the isolated NCl α chain monomer of type IV collagen isthe α1 chain monomer.
 22. The method of claim 19 wherein the isolatedNCl α chain monomer of type IV collagen is the α2 chain monomer.
 23. Themethod of claim 19 wherein the isolated NCl α chain monomer of type IVcollagen is the α3 chain monomer.
 24. The method of claim 19 wherein theisolated NCl α chain monomer of type IV collagen is the α6 chainmonomer.
 25. A method for inhibiting tumor metastasis in tissuecomprising contacting the tumor or tissue with an amount effective toinhibit tumor metastasis of a polypeptide composition comprising anisolated NCl hexamer of type IV collagen.
 26. A method for inhibitingtumor growth in tissue comprising contacting the tumor or tissue with anamount effective to inhibit tumor growth of a polypeptide compositioncomprising an isolated NCl hexamer of type IV collagen.
 27. A kit forpracticing the method of claim 25 comprising: a. an amount effective toinhibit tumor metastasis in an animal tissue of a polypeptidecomposition comprising an isolated NCl hexamer of type IV collagen, andb. instructions for using the amount effective of isolated NCl hexamerof type IV collagen to inhibit tumor metastasis in an animal tissue. 28.A kit for practicing the method of claim 26 comprising: a. an amounteffective to inhibit tumor growth in an animal tissue of a polypeptidecomposition comprising an isolated NCl hexamer of type IV collagen, andb. instructions for using the amount effective of isolated NCl hexamerof type IV collagen to inhibit tumor growth in an animal tissue.
 29. Amethod for treating an angiogenesis-mediated disease or condition in amammal, comprising administering to a mammal with anangiogenesis-mediated disease or condition an amount effective toinhibit angiogenesis of a polypeptide composition comprising one or moreisolated NCl α chain monomers of type IV collagen selected from thegroup consisting of α1, α2, α3, and α6 NCl chain monomers.
 30. Themethod of claim 29 wherein the isolated NCl α chain monomer of type IVcollagen comprises the α1 NCl chain monomer.
 31. The method of claim 29wherein the isolated NCl α chain monomer of type IV collagen comprisesthe α2 NCl chain monomer.
 32. The method of claim 29 wherein theisolated NCl α chain monomer of type IV collagen comprises the α3 NClchain monomer.
 33. The method of claim 29 wherein the isolated NCl αchain monomer of type IV collagen comprises the α6 NCl chain monomer.34. The method of claim 45 wherein the angiogenesis-mediated disease orcondition is selected from the group consisting of solid and blood-bornetumors, diabetic retinopathy, rheumatoid arthritis, retinalneovascularization, choroidal neovascularization, macular degeneration,corneal neovascularization, retinopathy of prematurity, corneal graftrejection, neovascular glaucoma, retrolental fibroplasia, epidemickeratoconjunctivitis, Vitamin A deficiency, contact lens overwear,atopic keratitis, superior limbic keratitis, pterygium keratitis sicca,sogrens, acne rosacea, phylectenulosis, syphilis, Mycobacteriainfections, lipid degeneration, chemical bums, bacterial ulcers, fungalulcers, Herpes simplex infections, Herpes zoster infections, protozoaninfections, Kaposi's sarcoma, Mooren ulcer, Terrien's marginaldegeneration, marginal keratolysis, traum, systemic lupus,polyarteritis, Wegeners sarcoidosis, scleritis, Steven's Johnsondisease, radial keratotomy, sickle cell anemia, sarcoid, pseudoxanthomaelasticum, Pagets disease, vein occlusion, artery occulsion, carotidobstructive disease, chronic uveitis, chronic vitritis, Lyme's disease,Eales disease, Bechets disease, myopia, optic pits, Stargarts disease,pars planitis, chronic retinal detachment, hyperviscosity syndromes,toxoplasmosis, post-laser complications, abnormal proliferation offibrovascular tissue, hemangiomas, Osler-Weber-Rendu, acquired immunedeficiency syndrome, ocular neovascular disease, osteoarthritis, chronicinflammation, Crohn's disease, ulceritive colitis, psoriasis,atherosclerosis, and pemphigoid.